Use of polyclonal human anti-HTG autoantibodies as a reagent for the clinical diagnosis of thyroid autoimmune diseases and reagent additive for detecting anti-HTG autoantibodies in patient sera

ABSTRACT

Use of polyclonal human autoantibodies against thyroglobulin (anti-hTg autoantibodies), in particular in the form of an affinity-purified IgG fraction, as a specific binding reagent in an immunological assay for the clinical detection of autoantibodies against thyroglobulin (anti-hTg autoantibodies) in the serum of a patient, in particular in competitive assays in which the anti-hTg autoantibodies to be determined and present in the sample and the polyclonal human anti-hTg autoantibodies used as the specific binding reagent compete for the binding sites of a human thyroglobulin (hTg) used as a further reagent of the assay, one of the stated reagents in each case being labelled and the other component in each case being bound to a solid phase, and in which the presence of the anti-hTg autoantibodies to be determined and present in the sample is detected on the basis of the reduction of the binding of the labelled reagent to the solid phase.

The present invention relates to the detection of antibodies inbiological fluids with the aid of immunological assays which are carriedout with the aid of reagent kits which are produced on an industrialscale and sold by producers of diganostic agents. In particular, thepresent invention relates to the detection of autoantibodies, inparticular autoantibodies against the autoantigens of the thyroid, inwhich in turn the determination of human autoantibodies againstthyroglobulin (hTg) in a human serum or plasma is of primary importancein connection with the present invention.

Thyroglobulin is one of the thyroid autoantigens and is a high molecularweight protein which consists of two identical glycosylated subunitshaving a molecular weight of about 330 kD each (cf. for example J.Furmaniak and B. Rees Smith in: Autoimmunity, 1990, Vol. 7, pages63-80). Thyroglobulin is a principle component of the thyroid colloidand is a precursor of the thyroid hormones triiodothyronine (T₃) andthyroxine (T₄). As a preliminary stage to the synthesis of the statedthyroid hormones, unbound circulating iodine or iodide is incorporatedin tyrosyl radicals or iodotyrosine radicals of the Tg under catalyticaction of the enzyme thyroidperoxidase (TPO). The thyroid hormones T₃and T₄ are then liberated from the iodinated thyroglobulin, this beingeffected by hormonal control mechanisms which respond to the content ofT₃ or T₄ in the blood.

In the case of thyroid autoimmune diseases, in particular in the case ofthe destructive Hashimoto's thyroiditis leading to hypothyroidism, inaddition to autoantibodies against thyroidperoxidase considerableamounts of autoantibodies against thyroglobulin are detected in thepatients' blood (such antibodies are always referred to below as"anti-hTg autoantibodies"). The detection and the quantitativedetermination of such anti-hTg autoantibodies are thus of considerableimportance in the clinical diagnosis of thyroid diseases.

The determination of anti-hTg autoantibodies in patient sera is possibleby various techniques, including immunological assays. In a commerciallyavailable assay of this type (HENNINGtest® anti-Tg), prediluted serumsamples are incubated in the test tubes simultaneously with a labelledTg (as tracer) and a protein A suspension. If anti-hTg autoantibodiesare present in the sample, they react with the antigen hTg added as thetracer. At the same time, the protein A present in the form of particlesof a suspended solid phase unspecifically binds to the Fc subunits ofthe antibodies, and a sandwich complex is formed. The particles of thesolid-phase suspension with all molecules bound thereto are convertedinto a sediment by centrifuging, and the supernatant containing theunbound tracer is removed. The larger the amount of autoantibodies inthe sample, the greater the amount of tracer which is bound. Theconcentration of the anti-hTg autoantibodies is thus directlyproportional to the amount of tracer detectable in the sediment, forexample to the radioactivity measurable in the sediment in the case ofradiolabelling.

By using the unspecific binding partner protein A, with the aid of whichwhole classes of IgG antibodies can be bound without distinction, and bythe simultaneous use of labelled thyroglobulin as a tracer, all anti-hTgautoantibodies are detected in the assay described. Since it is knownthat patient sera may also contain anti-hTg autoantibodies which cannotbe directly associated with a clinically manifest thyroid autoimmunedisease, assays which respond selectively to such anti-hTgautoantibodies which are directly linked with, and are the cause ofclinically manifest thyroid diseases, such as, for example, Hashimoto'sthyroiditis, would also be of interest per se. Moreover, the fact thatthe known method described requires a centrifuging step may be regardedas a certain disadvantage of said method. However, another method whichworks as a competitive assay and, as a coated tube method, manageswithout a centrifuging step (DYNOtest® anti-TPO; cf. DE 41 20 412 C1with regard to the principle of the method) is available for thedetermination of anti-TPO autoantibodies, which is very frequentlycarried out parallel to the determination of anti-hTg autoantibodies.However, simultaneously carrying out two assays which involve differenthandling steps proves to be a disadvantage in clinical laboratorypractice. It would therefore be desirable per se also to be able tocarry out the determination of anti-hTg autoantibodies by an assay whichworks according to the coated tube technique and is a competitive assayand in which the presence of the antibodies to be determined in theinvestigated serum sample manifests itself as a reduction of the bindingof the labelled reagent (tracer) to the walls of a test tube.

While the epitopes of the antigen hTPO are essentially known in the caseof the determination of anti-TPO autoantibodies and it is possible tofind a set of monoclonal antibodies which are suitable for a competitivedetermination (cf. Jean Ruf, Marie-Elisabeth Toubert, Barbara Czarnocka,Josee-Martine Durand-Gorde, Mireille Ferrand and Pierre Carayon, in:Endocrinology, Vol. 125, No. 3, pages 1211-1218, 1989), the developmentof comparable competitive assays has to date come up against fundamentalproblems in cases where the antigens are very large and/orinsufficiently characterized with regard to their epitopes involved inthe immunoreaction or where said epitopes are even unknown. Such casesare very numerous, and glutamate decarboxylase (diabetes type I), theacetylcholine receptor (myasthenia gravis), the so-called "myelin basicprotein" (multiple sclerosis), the TSH receptor and in particularthyroglobulin (hTg) may be mentioned as examples of such antigens.

If an attempt is made to develop, for the determination of anti-hTGautoantibodies, a competitive assay which corresponds to the methoddescribed above for the determination of anti-TPO autoantibodies,difficulties are encountered in connection with the peculiarities of thethyreoglobulin acting as an antigen or the antibody populations formedagainst thyroglobulin. As mentioned at the outset, human thyroglobulin(hTg) is a very large protein antigen having a very large number ofpotential antigen determinants (epitopes) which, with the use of humanthyroglobulin for producing heterologous antibodies by directimmunization of an animal, result in a population of polyclonalantibodies being obtained which were found to be directed against alarge number of different epitopes of human thyroglobulin. However,investigations in recent years have shown that the anti-hTgautoantibodies formed during autoimmune diseases are evidently formedonly against a limited number of epitopes on the thyroglobulin surface(J. Furmaniak and B. Rees Smith, in: Autoimmunity, 1990, Vol. 7, pages63-80; J. Ruf, Mireille Henry, Catherine De Micco, P. Carayon, in:Thyroglobulin and Thyroglobulin Antibodies in the Follow-up of ThyroidCancer and Endemic Goiter (Hufner, M., and Reiners, C., Editors), pages21-31, 1987, G. T. Verlag Stuttgart, New York; C. T. J. Chan, P. G. H.Byfield, R. L. Himsworth and P. Shepherd, in: Clin.exp.Immunol. (1987)70, pages 516-523; S. M. McLachlan, U. Feldt-Rasmussen, E. T. Young, S.L. Middleton, M. Blichert-Toft, K. Siersboek-Nielsen, J. Date, D. Carr,F. Clark and B. Rees Smith, in: Clinical Endocrinology (1987), 26, pages335-346; Q. Dong, M. Ludgate and G. Vassart, in: Journal ofEndocrinology (1989) 122, pages 169-176; M. E. Devey, K. M.Bleasdale-Barr, S. M. McLachlan, J. Bradbury, F. Clark and E. T. Young,in: Clin.exp.Immunol. (1989), 77, pages 191-195; N. Fukuma, S. M.McLachlan, V. B. Petersen, P. Kau, J. Bradbury, M. Devey, K. Bleasdale,P. Grabowski and B. Rees Smith, in: Immunology 1989, 67, pages 129-131;N. Fukuma, S. M. McLachlan, V. B. Petersen, K. Beever and B. Rees Smith,in: Autoimmunity, 1990, Vol. 6, pages 37-45; Shannon L. Gleason,Patricia Gearhardt, Noel R. Rose and Rudolf C. Kuppers, in: The Journalof Immunology, Vol. 145, No. 6, pages 1768-1775, 1990; Herbert S.Bresler, C. Lynne Burek and Noel R. Rose, in: Clinical Immunology andImmunopathology, Vol. 54, pages 64-75, (1990); Herbert S. Bresler, C.Lynne Burek, William H. Hoffman and Noel R. Rose, in: ClinicalImmunology and Immunopathology, Vol. 54, pages 76-86, (1990); MireilleHenry, Yves Malthiery, Eric Zanelli and Bernadette Charvet, in: TheJournal of Immunology, Vol. 145, No. 11, pages 3692-3698, 1990; ShigekiSakata, Toru Ogawa, Yasuyoshi Kimata, Hiroshi Takuno, Hiroshi Maekawa,Masafumi Matsuda, Osamu Tarutani and Kenji Okuda, in: Molecular andCellular Endocrinology, 79, (1991), pages 93-98; Gilles Dietrich,Martine Pierchaczyk, Bernard Pau and Michel D. Kazatchkine, in: Eur. J.Immunol. 1991, 21, pages 811-814; Yves Malthiery, Mireille Henry andEric Zanelli, in: FEBS Letters, Vol. 279, No. 2, pages 190-192 (1991);E. Schulz, G. Benker, H. Bethauser, L. Stempka and M. Hufner, in: J.Endocrinol. Invest 15, pages 25-30, 1992; Asmae Alami Harchali, PaulMontagne, Marie L. Cuilliere, Majida Bouanani, Bernard Pau and JeanDuheille, in: Clin. Chem. Vol. 38, No. 9, pages 1859-1864 (1992); B.Mallet, P. J. Lejeune, J. Ruf, M. Piechaczyk, C. Marriq and P. Carayon,in: Molecular and Cellular Endocrinology, 88, (1992), pages 89-95; J. M.Hexham, J. Furmaniak, C. Pegg, D. R. Burton and B. Rees Smith, in:Autoimmunity, 1992, Vol. 12, pages 135-141; Rudolf C. Kuppers, Ingrid M.Outschoorn, Robert G. Hamilton, C. Lynne Burek and Noel R. Rose, in:Clinical Immunology and Immunopathology, Vol. 67, No. 1, pages 68-77,1993; Peter J. Delves, Sandra M. McLachlan, Elizabeth Drewe, Nao Fukuma,Vaughan B. Petersen and Bernard Rees Smith, in: Journal of Autoimmunity(1993), 1, pages 77-91; Patrizio Caturegli, Stefano Mariotti, Rudolf C.Kuppers, C. Lynne Burek, Aldo Pinchera and Noel R. Rose, in:Autoimmunity, 1994, Vol. 18, pages 41-49). Owing to the complexity ofthe problem, complete epitope mapping has to date been unsuccessful forhTg, the situation being made more difficult by the fact that it hasbeen found that the autoantibodies formed during autoimmune diseases areapparently among the conformative antibodies, which makes the attempt atepitope mapping with the use of monoclonal anti-hTg antibodies of animalorigin even more difficult and prevents mapping of the relevant epitopeswith the use of recombinant, sequential antibodies (Q. Dong, M. Ludgateand G. Vassart, in: Journal of Endocrinology (1989) 122, pages 169-176).Polyclonal heterologous IgG populations obtained by immunization withthe aid of hTg in the sera of animals thus do not correspond to theanti-hTg autoantibody populations occurring in autoimmune diseases andare therefore evidently not very suitable or unsuitable for use incompetitive immunological assays.

On the other hand, it has been found that the attempt to use monoclonalanti-hTg antibodies in competitive assays for determining anti-hTgautoantibodies also does not lead to the desired results, namely thedevelopment of an assay which determines the proportion of thoseanti-hTg autoantibodies which are actually associated with thepathological process of an autoimmune disease. The reason for theunsatisfactory results with the use of monoclonal anti-hTg antibodiespresumably lies in the fact that the individual epitopes which areinvolved in the anti-hTg autoantibody formation influence one another tosuch a small extent on the very large hTg molecule that only fractionsof the polyclonal anti-hTg autoantibodies actually present in thepatient serum are detected in competitive assays using monoclonalanti-hTg antibodies, and that these fractions do not adequatelyrepresent the total pathological process.

Attempts to use monoclonal or recombinant human anti-hTg antibodiesinstead of monoclonal anti-hTg antibodies of animal origin (cf. GB-A-2265 713) do not in principle alter this problem.

It has thus been found that the determination of anti-hTg autoantibodieson the basis of existing competitive assays is unreliable and cannot becarried out with sufficient sensitivity.

It is therefore the object of the present invention to designcompetitive assays of antibodies, in particular competitive assays forthe determination of anti-hTg autoantibodies, in such a way that suchautoantibodies can be determined with high accuracy and reliability evenwhen these autoantibodies are formed against antigens having a largenumber of relevant epitopes and are present as a population ofpolyclonal antibodies.

This object is achieved in the determination of anti-hTg autoantibodiesin patient sera according to the invention by the use of polyclonalhuman autoantibodies against thyroglobulin as a specific bindingreagent, in particular affinity-purified polyclonal human IgGautoantibodies against thyroglobulin from sera of human patients whosuffer from a thyroid autoimmune disease.

Particularly advantageous methods of the use, according to theinvention, of polyclonal anti-hTg autoantibodies are describedhereinbelow.

The present invention furthermore relates to a reagent kit for theimmunological determination of anti-hTg autoantibodies in patient sera,which is essentially characterized in that it contains affinity-purifiedanti-hTg autoantibodies (an affinity-purified anti-hTg IgG preparation)in addition to conventional components of such a reagent kit.

In accordance with a very much more general aspect which describedhereinbelow, the present invention furthermore relates, still generally,to the use of polyclonal human antibodies for the clinical diagnosis ofdiseases which are characterized by the occurrence of such antibodiessince, as will be explained in more detail, the principles arising outof the determination of anti-hTg autoantibodies are applicable to otherapplications and it was not possible to establish that polyclonal humanantibodies (hIgGs) obtained from the sera of sufferers have already beenused as specific binding reagents in commercial reagent kits forcarrying out immunological assays.

Where it is stated in the present application that polyclonal humanautoantibodies against thyreoglobulin are used as a "specific bindingreagent", this means that they are used as reactants in an immunologicalassay in which a specific immunological reaction between antigen andantibody or antibodies is utilized. "Specific binding reagent" is notused in the meaning of "binder for analytes", since it would benecessary to regard as "analytes" the antibodies to be determined andwith which the polyclonal human autoantibodies against thyroglobulin,which are to be used according to the invention, compete as competitorsfor binding sites of the antigen thyroglobulin.

The present invention is based on the knowledge that it is possible veryreliably to detect anti-hTg autoantibodies in the serum of a patient if,instead of using antibodies (polyclonal or monoclonal anti-hTgantibodies) of animal origin, as has been usual to date in the area ofthe industrial preparation of reagent kits for immunological assays,polyclonal human autoantibodies which are obtained directly from theserum of patients shown to be suffering from the thyroid autoimmunedisease to be detected are used. In the present invention, it ispossible in general to use such polyclonal human autoantibodies in theform of a serum of an individual patient with the typical symptoms ofthe thyroid autoimmune disease to be detected, but it is preferable touse the combined ("pooled") sera of a plurality of such patients as asource for the polyclonal human autoantibodies, in order to ensure thatthe starting serum used contains as complete a population as possible ofall autoantibodies which may be formed in humans in connection with thedisease to be detected. Furthermore, in the case of the determination ofanti-hTg autoantibodies, the polyclonal human autoantibodies arepreferably not used in such a way that all IgG autoantibodies present insuch a serum are employed; instead, the polyclonal human autoantibodiesare preferably used, after affinity purification with the use of a hTgaffinity matrix, in such a way that the immunoglobulin fraction used asa specific binding reagent comprises essentially only anti-hTgautoantibodies.

Where the present application refers to the use of thyroglobulin orhuman thyroglobulin for the purpose of the affinity purification or as areagent in an immunological assay, what is primarily meant is aconventional human thyroglobulin, as is commercially available fromvarious producers, for example from the British company Scipac. However,since it has recently been shown that very specific thyroglobulinfractions may be involved to an above-average extent in the formation ofautoantibodies during thyroid autoimmune diseases (Ali M. Saboori, NoelR. Rose, Rudolf C. Kuppers, Wayne G. Butscher, Herbert S. Bresler and C.Lynne Burek, in: Clinical Immunology and Immunopathology, Vol. 72, No.1, pages 121-128, 1994; A. Gardas, in: Autoimmunity, 1991, pages331-336), the present invention expressly uses, both for affinitypurification and as a reagent in the assay, not a complete thyroglobulinpreparation but only a human thyroglobulin fraction which isparticularly important for autoimmune diseases, for example the proteinfraction which is designated as protein peak 1 in the stated publicationand which corresponds to a thyroglobulin having a greatly reduced degreeof iodination, or another fraction of hTg, which can be shown to occurto a greater extent, for example, in those suffering from Hashimoto'sthyroiditis than in healthy persons. By using such a thyroglobulinfraction instead of a complete thyroglobulin preparation, it may onceagain be possible to achieve further advantages with regard to theselectivity and/or sensitivity and specificity of the assay.

The determination, according to the invention, of human anti-hTgautoantibodies is illustrated in more detail below with reference to twoFigures and two measured curves and two Use Examples.

In the Figures,

FIG. 1 shows the principle of the method in a first variant of ananti-hTg autoantibody determination using an affinity-purifiedpolyclonal human IgG fraction which serves as an immobilized specificbinder for the hTg used in labelled form;

FIG. 2 shows the principle of the method in a second variant of ananti-hTg autoantibody determination using an affinity-purifiedpolyclonal human IgG fraction which is used in labelled form as aspecific binder for the hTg used in immobilized form;

FIG. 3 shows a typical measured curve with associated table of measuredvalues for a determination of anti-hTg autoantibodies in an assayaccording to Example 1; and

FIG. 4 shows a typical measured curve with associated table of measuredvalues for a determination of anti-hTg autoantibodies in an assayaccording to Example 2.

By the use of polyclonal human anti-hTg autoantibodies as specificbinding reagents, it is possible to realize competitive immunologicalassays in which the polyclonal human autoantibodies added as specificbinding reagents compete with the autoantibodies to be detected in thepatient's sample.

In particular, the assay can be carried out in such a way that (cf. FIG.1), according to one variant, the polyclonal human anti-hTgautoantibodies, in particular in the form of an affinity-purifiedanti-hTg-IgG preparation, bind to a solid carrier phase, in particularto the wall of a test tube, and that a labelled human thyroglobulin, forexample a radioiodinated human thyroglobulin, is used as a furtherreagent. In the absence of anti-hTg autoantibodies, in particular in theabsence of hTg autoantibodies which are the cause of the thyroid diseaseto be detected, the labelled thyroglobulin is bound completely or to apercentage characteristic of a blank sample. If anti-hTg autoantibodiesof the type to be detected are present in the patient's sample, theresult is competition between the antibodies used in the method as aspecific binding reagent and the antibodies in the patient's serum,which is manifested in reduced binding of the labelled thyroglobulin tothe solid phase, which, after a solid/liquid separation, results in lesslabel being found on the solid phase.

In another principle of the method which is also known per se inprinciple (FIG. 2), it is also possible to follow a procedure in whichthe antigen, that is to say human thyroglobulin (or a fraction thereof,see above), is immobilized on a solid phase and a tracer in the form ofa labelled polyclonal human anti-hTg autoantibody fraction is used. Inthis case too, the presence of the autoantibodies to be determined in apatient's sample leads to a reduction in the binding of the label to thesolid phase.

Even if an iodine radioisotope is always used in the Examples of thepresent invention as a marker or label for the tracer molecule, it isevident to a person skilled in the art that the nature of the label usedis not critical and any other suitable label can be chosen and used fortracer preparation.

The solid phase may also be the wall of a microtitre plate or anothercoarsely disperse or finely disperse solid phase known per se, and thesuitable solid phase can be chosen primarily on the basis of practicalconsiderations for the particular application.

As the tests below show, it has surprisingly been found that a reliableanti-hTg autoantibody determination in human sera is possible bycompetitive assay with the use of polyclonal human anti-hTgautoantibodies as a specific binding reagent.

The use of polyclonal human anti-hTg autoantibodies as a specificbinding reagent presumably has the advantage that binding partners forall possible epitopes which are associated with the pathogenicdevelopment of anti-hTg autoantibodies are present on the hTg moleculein such a polyclonal specific binding reagent. However, this ensuresthat any occurrence of such anti-hTg autoantibodies in a patient's serumleads to a detectable effect, the effect of course increasing with theamount and/or the number of individual autoantibody types in a patient'sserum. The possibility, with the use of monoclonal antibodies as aspecific binding reagent, that in fact the specific binding betweenmonoclonal antibody and antigen will not be disturbed by a certain typeof autoantibody from a patient's serum and such autoantibodies will thusnot be detected is not expected in the method according to theinvention. By using patients' sera having autoantibodies which are atypical set for a certain disease and, as an antibody population,represent a sort of fingerprint distribution which can be correlatedwith a certain pathological condition, the optimum possible specificityfor such a fingerprint pattern is furthermore obtained, and unspecificside effects can be eliminated by suitable standardization.

When polyclonal human anti-hTg autoantibodies are used, there issufficient probability that--even with immobilization or labelling ofthese antibodies or of the associated antigens--all relevant epitopes ofhTg are available for binding with all relevant autoantibody types, sothat each individual antibody type of the population can make acontribution to the total measured result, the clinical relevance ofwhich is thus increased.

However, a precondition for the present invention was also thesurprising discovery that there is in practice no serious problem inworking with antibodies obtained from sera of human donors sufferingfrom a disease instead of with antibodies obtained by immunization ofanimals or with corresponding monoclonal antibodies, as has been usualto date. It was surprisingly found that, for example, anti-hTgautoantibodies are present in sera of patients with Hashimoto'sthyroiditis in amounts such that a relatively small amount of donorserum is sufficient to meet the requirement for the preparation of alarge amount of coated test tubes. Thus, 5 mg of affinity-purifiedanti-hTg autoantibodies, which is sufficient for about 25000 individualdeterminations, can be obtained, for example, using 10 ml of patient'sserum with a content of, for example, 10000 U/ml of anti-Tg (determinedin the HENNINGtest® anti-Tg of the Applicant). Furthermore, it hassurprisingly been found that, when pooled sera of a sufficient,relatively small number of different persons suffering from a diseaseare used, very suitable polyclonal human autoantibodies withsubstantially identical binding properties are obtained for the intendeduse, and relative fluctuations which may be observed from batch to batchcan easily be taken into account by means of the envisaged usualcalibration steps.

Reservations with regard to adequate availability and/or reproducibilityand constancy of quality of the specific binding reagents are thereforeunjustified in assays with the use of polyclonal human antibodies. Suchreservations--amounting to a prejudice--might explain why human IgGfractions are at present never used in the commercial assays as specificbinding partners and why it was also not possible to find in theliterature a proposal for the use of polyclonal human IgG fractions asspecific binding partners for the preparation of commercial reagent kitsfor immunological assays for the determination of antibodies inpatients' sera.

It is evident from the above statements, in particular with regard tothe detection specificity discussed, which is capable of detecting the"fingerprint pattern" of disease-specific antibody populations, that theprinciple on which the present invention is based is suitable not onlyfor the determination of anti-hTg autoantibodies but generally forantibody determination in clinical diagnosis, and in particular also forthe determination of antibodies against external antigens. A possibleapplication of this type consists, for example, in using IgG fractionsobtained from an individual patient while suffering from his disease asspecific binding partners for assays which are used for monitoring theimprovement in the condition of this specific patient on the basis ofthe reduction in the antibodies circulating in the patient's blood. Insuch a case, a doctor can be provided with a reagent kit which containsall required reagents except for the test tubes to be coated by thedoctor himself with an IgG fraction obtained from the patient. If thedoctor prepares a certain stock of such patient-specific test tubes, heis able subsequently to monitor the changes in the antibodyconcentrations in a patient and possibly the disappearance of theoriginal antibodies.

Similarly, human antibodies against a certain antigen from patients'sera, which have been pooled and if necessary concentrated by affinitypurification, can be used to prepare a test system which makes itpossible to determine, in a general but clinically highly relevantmanner, the immune status of patients who are suspected of sufferingfrom a disease caused by this antigen. Suitable antigens are not onlyautoantigens but also pathogenic antigens of bacterial or viral originor antigens which act as allergens. Such test systems may even beprovided when very little is known about the antigens and the type ofantibodies formed against these antigens, since the test system to acertain extent directly indicates the presence or absence of acorresponding pathogenic condition, provided that this is reflected in areaction of the immune system with adequate antibody formation.

The present invention is illustrated in more detail below with referenceto two examples for the two assay types for anti-hTg autoantibodiesshown in the Figures. However, it should also be noted at this pointthat the principle of the present invention has also proved its worth inother cases where, as in the case of the determination of anti-hTPOautoantibodies, competitive immunological assays with the use ofmonoclonal antibodies give essentially correct results in thepredominant number of cases. With the use of polyclonal human antibodiesin the form of an IgG fraction, it was possible to obtain comparablecorrect results in this case even without prior affinity purification;moreover, in the very rare cases of sera incorrectly determined asantibody-free, the correct result was obtained, indicating that the seradid in fact contain the autoantibodies being searched for.

EXAMPLES

Materials

The tubes used as solid phases in all Examples are tubes from Nunc,Maxisorp quality, 75×12 mm, with star insert.

The buffer solutions mentioned are:

Buffer A: Phosphate-buffered saline solution (PBS), 0.1% Tween 20

Buffer B: 10 mM TRIS HCl, 10 mM NaCl, pH 7.8

Buffer C: 20 mM sodium phosphate, pH 7.0

Buffer D: 25 mM citric acid

Buffer E: 0.5 M sodium phosphate, pH 8.0

Buffer F: 20 mM sodium phosphate, 1% bovine serum albumin (BSA; Sigma),3% crystallization-inhibited sorbitol syrup (Karion FP; Merck), pH 7.5

Serum samples:

Serum samples from patients with the indications Hashimoto'sthyroiditis, autoimmune thyroiditis and Graves' Disease were obtainedfrom various German hospitals. They were used for the preparation of thereagents or as test sera. Serum samples which are used as controlsamples are sera from persons who were without symptoms of anyinfections, autoimmune diseases or allergic diseases.

The assay according to HENNINGtest® anti-Tg, used as a comparativemethod, is a commercial assay and is available in the form of a reagentkit with working instructions from BRAHMS Diagnostica GmbH, Berlin.

Iodine¹²⁵ -labelled human thyreoglobulin was taken from the statedcommercial HENNINGtest® anti-Tg.

Affinity purification of anti-hTg autoantibodies from sera of patientswith Hashimoto's thyroiditis for preparation of reagents

a) Preparation of an affinity matrix

20 mg of human thyroglobulin (Scipac, GB) are dissolved in 7 ml ofbuffer C, and 100 ml of sodium periodate (Fluka) are added. Afterincubation for 15 minutes, the solution is desalted by means of NAP 25columns (Pharmacia) and then mixed with 10 ml of Carbolink material(Pierce, USA). After incubation for 20 hours at 4° C., the material isintroduced into a 10 ml glass column (Biorad) and washed with buffer C.

b) Affinity purification of the human anti-Tg autoantibodies from seraof Hashimoto patients

1 ml of serum from each of 10 patients with Hashimoto's thyroiditis aremixed (final anti-hTg content corresponding to a determination withHENNINGtest® anti-Tg of the Applicant: 5000 U/ml) and are circulated for120 min over the affinity matrix using a peristaltic pump at a flow rateof 1 ml/min. The absorbance of the column outflow is measuredcontinuously at 280 nm. The column is then washed with 50 ml of bufferC.

The human anti-hTg autoantibodies are eluted by washing the column withbuffer D. The human anti-hTg autoantibodies (volume after elution: 4 ml)are mixed with 4 ml of buffer E and stored at 4° C. until required forfurther use.

Example 1

Determination of anti-hTg autoantibodies using a radioiodinatedthyreoglobulin as tracer (method HD-R):

a) Preparation of test tubes with human antibodies from patients withHashimoto's thyroiditis ("HD-R tubes")

Tubes which were coated with anti-human-IgG for binding the humanantibodies were first prepared. For this purpose, goat-anti-human-IgG(Order No. 3AG474, Grade 2, from Scantibodies, USA) was diluted inbuffer B to a final concentration of 6.7 μg/ml. 300 μl of this solutionwere pipetted in test tubes (Maxisorp tubes from Nunc) and incubated for20 h at room temperature. The tube content was then decanted, and thetubes were completely filled with buffer F. After incubation for 2hours, the tube content was decanted and the tubes were dried in vacuo.The prepared tubes ("AHI tubes") were stored at 4° C.

The human anti-hTg autoantibodies affinity-purified as described abovewere diluted with buffer A to a protein concentration of 2 μg/ml, and100 μl of this solution were pipetted into each of the tubes coated withanti-human-IgG (AHI tubes) and incubated for 2 hours with shaking (at320 revolutions/min) at room temperature. A solution containing 1 mg/mlof human IgG in buffer A is then pipetted to saturate residual freeanti-human-IgG binding sites and incubation is continued for a further 2h with shaking. The tubes are then washed twice with 2 ml of PBS eachtime and are stored at 4° C. until required for further use.

b) Carrying out the determination

The procedure was based on the following incubation scheme:

1. For the preparation of the samples, dilute human serum of the controlgroups, and of the patients and of the calibrators in a ratio of 1+20(one part of serum, 20 parts of buffer) with buffer A.

2. Pipette 100 μl of the sample into the tubes coated with antibodies ofpatients with Hashimoto's thyroiditis (HD-R tubes).

3. Pipette 100 μl of a radioiodinated human Tg (component of thecommercial HENNINGtest® anti-Tg assay).

4. Incubate for 2 hours at room temperature with shaking.

5. Remove the liquid phase and wash twice with 2 ml of PBS each time.

6. Measure the radioactivity remaining in the tubes.

c) Results

In the absence of anti-thyroglobulin autoantibodies in the sample underthe stated conditions, the radioiodinated thyroglobulin tracer is boundto an extent of 73% to tubes which are coated with IgG from patientswith Hashimoto's thyroiditis. Owing to increasing amounts of anti-Tgautoantibodies in the samples, the binding of the labelled thyroglobulindecreases. Typical measured values of the calibration and a typicalmeasured curve are shown in FIG. 3.

As expected, 41 control samples are found to be negative in the methoddescribed, exactly as in the comparative method HENNINGtest® anti-Tgfrom BRAHMS Diagnostica (cf. Table 1).

85 sera from patients with either Hashimoto's thyroiditis, Graves'Disease or immune thyroiditis were measured in the method according tothe invention and in the comparative method HENNINGtest® anti-Tg.

Of the measured samples, which are potentially anti-hTg-positive, 35samples are found to be positive in the method according to theinvention and 32 in the comparative method. 28 samples are found to bepositive in both methods. The measured results are listed in Table 2.The results show that the use of polyclonal human anti-hTgautoantibodies is suitable for the determination of autoantibodiesagainst thyreoglobulin in patient sera.

Example 2

Determination of anti-hTg autoantibodies with the use of labelledaffinity-purified human anti-hTg autoantibodies from Patient sera(method Tg-R)

a) Radioiodination of human anti-hTg autoantibodies

10 μg of the human anti-hTg autoantibodies affinity-purified asdescribed above and obtained from the sera of patients suffering fromHashimoto's disease, in a volume of 50 μl, are mixed with 50 μl ofsodium phosphate buffer, 50 mM, pH 7.4, and 150 μCiNal¹²⁵ I in 2 μl, andthe labelling reaction is carried out by the classical Chloramine Tmethod and started by the addition of 2.5 μg of Chloramine T in 5 μl ofH₂ O. After a reaction time of 60 seconds, the sample is diluted with300 μl of 50 mM sodium phosphate buffer pH 7.4 and desalted by means ofan NAP-10 column (Pharmacia) in the usual manner as prescribed. Thelabelled antibody obtained is diluted with 50 mM sodium phosphatebuffer, 0.1% Tween 20, 0.2% BSA, pH 7.4, to a final dilution of 400,000cpm/ml. The material is stored at 4° C.

b) Procedure

For the determination, the procedure is based on the followingincubation protocol:

1. For sample preparation, dilute human serum from control groups, frompatients and from calibrators 1+20 with buffer A.

2. Pipette 100 μl of the sample into test tubes coated withthyroglobulin

3. Pipette 100 μl of the radioiodinated human anti-hTg antibody preparedas above

4. Incubate for two hours at room temperature with shaking

5. Separate off the liquid phase and wash the tubes twice with 2 ml ofPBS each time

6. Measure the radioactivity remaining in the tubes.

c) Results

In the absence of anti-hTg antibodies in the sample under the statedconditions, the radioiodinated human anti-hTg antibody is bound to anextent of 32% to tubes which are coated with human thyroglobulin (Tg-R).Owing to the increasing amounts of anti-hTg antibodies in the sample,the binding of the labelled human anti-hTg antibody decreases (a typicalmeasured curve is shown in FIG. 4).

As expected, 10 control samples (cf. Table 1) are found to be negativein the method described, as in the comparative method HENNINGtest®anti-Tg from BRAHMS Diagnostica GmbH.

In the measurement of the sera of 85 patients with Hashimoto'sthyroiditis, Graves' Disease or immunethyroiditis, 16 samples weredetermined as being positive by means of the present method. In thecomparative method HENNINGtest® anti-Tg, 17 samples were determined asbeing positive. 14 samples of the present variant of the assay were inagreement with the comparative method HENNINGtest® anti-Tg (cf. Table2).

It is thus found that it is also possible to use human anti-hTgantibodies obtained from patient sera and present in labelled form forthe determination of corresponding autoantibodies against thyroglobulinin patient sera.

                  TABLE 1    ______________________________________    Measurement of control sera    Control group          Immu anti-Tg      HD-R         Tg-R    No.   U/ml              U/ml         U/ml    ______________________________________     1    <100       neg.   <30     neg. <60    neg.     2    <100       neg.   <30     neg. <60    neg.     3    <100       neg.   <30     neg. <60    neg.     4    <100       neg.   <30     neg. <60    neg.     5    <100       neg.   <30     neg. <60    neg.     5    <100       neg.   <30     neg. <60    neg.     7    <100       neg.   <30     neg. <60    neg.     8    <100       neg.   <30     neg. <60    neg.     9    <100       neg.   <30     neg. <60    neg.    10    <100       neg.   <30     neg. <60    neg.    11    <100       neg.   <30     neg.    12    <100       neg.   <30     neg.    13    <100       neg.   <30     neg.    14    <100       neg.   <30     neg.    15    <100       neg.   <30     neg.    16    <100       neg.   <30     neg.    17    <100       neg.   <30     neg.    18    <100       neg.   <30     neg.    19    <100       neg.   <30     neg.    20    <100       neg.   <30     neg.    21    <100       neg.   <30     neg.    22    <100       neg.   <30     neg.    23    <100       neg.   <30     neg.    24    <100       neg.   <30     neg.    25    <100       neg.   <30     neg.    26    <100       neg.   <30     neg.    27    <100       neg.   <30     neg.    28    <100       neg.   <30     neg.    29    <100       neg.   <30     neg.    30    <100       neg.   <30     neg.    31    <100       neg.   <30     neg.    32    <100       neg.   <30     neg.    33    <100       neg.   <30     neg.    34    <100       neg.   <30     neg.    35    <100       neg.   <30     neg.    36    <100       neg.   <30     neg.    37    <100       neg.   <30     neg.    38    <100       neg.   <30     neg.    39    <100       neg.   <30     neg.    40    <100       neg.   <30     neg.    41    <100       neg.   <30     neg.    ______________________________________

                  TABLE 2    ______________________________________    Measurement of patient sera    Patient sera with following diagnoses: Hashimoto, Graves' disease,    immune thyroiditis          Henningtest anti-Tg HD-R        Tg-R    No.   U/ml                U/ml        U/ml    ______________________________________     1    <100         neg.   <30    neg. <80   neg.     2    2156         pos.   1350   pos. 685   pos.     3    2020         pos.   1747   pos. 850   pos.     4    1148         pos.   <30    neg. <80   neg.     5     221         pos.   422    pos. 174   pos.     6    1248         pos.   1434   pos. 820   pos.     7    4155         pos.   4208   pos. 4150  pos.     8    1030         pos.   870    pos. 794   pos.     9    2568         pos.   2940   pos. 1849  pos.    10    2338         pos.   2520   pos. 1644  pos.    11    <100         neg.   <30    neg. <60   neg.    12     107         pos.   158    pos. <60   neg.    13    4416         pos.   3910   pos. 4635  pos.    14    1576         pos.   170    pos. 144   pos.    15     972         pos.   973    pos. 620   pos.    16    <100         neg.   <30    neg. 333   pos.    17    <100         neg.    77    pos. <60   neg.    18    2467         pos.   701    pos. 441   pos.    19    <100         neg.   <30    neg. <60   neg.    20    <100         neg.   <30    neg. <60   neg.    21    <100         neg.   <30    neg. <60   neg.    22    <100         neg.   <30    neg. 407   pos.    23    <100         neg.   <30    neg. <60   neg.    24     963         pos.   <30    neg. <60   neg.    25    <100         neg.   <30    neg. <60   neg.    26     170         pos.   345    pos.  84   pos.    27     724         pos.   111    pos. 134   pos.    28    <100         neg.   <30    neg.    29    <100         neg.   <30    neg.    30    <100         neg.   <30    neg.    31    <100         neg.   <30    neg.    32    2122         pos.   223    pos.    33     952         pos.   <30    neg.    34     567         neg.    94    pos.    35     221         neg.   318    pos.    36    <100         pos.   <30    neg.    37    <100         neg.   <30    neg.    38    5139         pos.    66    pos.    38    <100         neg.   335    neg.    40    <100         neg.   <30    neg.    41    <100         neg.   <30    neg.    42    <100         neg.    84    neg.    43     322         pos.   <30    pos.    44    <100         neg.   <30    neg.    45    <100         neg.   <30    neg.    46    <100         neg.   <30    neg.    47    <100         neg.   <30    neg.    48    <100         neg.   <30    neg.    49    <100         neg.    46    pos.    50    <100         neg.   <30    neg.    51    <100         neg.   139    pos.    52    16443        pos.   17173  pos.    53     645         pos.   413    pos.    54    <100         neg.   <30    neg.    55    <100         neg.   <30    neg.    56    <100         neg.   <30    neg.    57    <100         neg.   <30    neg.    58    <100         neg.   <30    neg.    59    <100         neg.   <30    neg.    60    <100         neg.   <30    neg.    61    <100         neg.   <30    neg.    62    <100         neg.   <30    neg.    63    <100         neg.    69    pos.    64    <100         neg.   <30    neg.    65    <100         neg.   <30    neg.    66    3449         pos.   3796   pos.    67    <100         neg.   <30    neg.    68    971          pos.   <30    neg.    69    <100         neg.   <30    neg.    70    <100         neg.    90    pos.    71    <100         neg.   <30    neg.    72    <100         neg.    83    pos.    73    <100         neg.   <30    neg.    74     274         pos.   207    pos.    75     156         pos.   241    pos.    76    <100         neg.   <30    neg.    77     976         pos.    42    pos.    78    <100         neg.   <30    neg.    79    <100         neg.   <30    neg.    80    <100         neg.   <30    neg.    81    <100         neg.   <30    neg.    82     822         pos.   608    pos.    83     978         pos.   285    pos.    84    <100         neg.   <30    neg.    85    <100         neg.   <30    neg.    ______________________________________

I claim:
 1. A method for detection of anti-human thyroglobulin(anti-hTg) autoantibody in a fluid test sample, which methodcomprises:(A) combining:said test sample, a first reagent comprisingpolyclonal human anti-hTg autoantibodies, and a second reagentcomprising a human thyroglobulin (hTg) preparation,wherein one of saidfirst reagent and said second reagent is a labeled reagent and the otherof said first reagent and said second reagent is an immobilized reagentbound to a solid phase; under conditions such thatanti-hTg autoantibodyin said test sample, if any, binds to hTg in said hTg preparation,thereby reducing the extent of binding of hTg to said polyclonal humananti-hTg autoantibodies in said first reagent compared to the extent ofbinding of hTg to said polyclonal human anti-hTg autoantibodies in saidfirst reagent in the absence of said sample, (B) determining the amountof said labeled reagent immobilized on said solid phase by saidimmobilized reagent, and (C) correlating the amount of said labeledreagent immobilized by said immobilized reagent, if any, in the presenceof said test sample with the amount of said labeled reagent immobilizedby said immobilized reagent under the same conditions in the absencesaid test sample, whereby anti-hTg autoantibody in said sample, if any,is detected.
 2. A method for determining the amount of anti-humanthyroglobulin (anti-hTg) autoantibody in a fluid test sample, whichmethod comprises:(A) combining:said test sample, a first reagentcomprising polyclonal human anti-hTg autoantibodies, and a secondreagent comprising a human thyroglobulin (hTg) preparation,wherein oneof said first reagent and said second reagent is a labeled reagent andthe other of said first reagent and said second reagent is animmobilized reagent bound to a solid phase; under conditions suchthatanti-hTg autoantibody in said test sample, if any, binds to hTg insaid hTg preparation, thereby reducing the extent of binding of hTg tosaid polyclonal human anti-hTg autoantibodies in said first reagentcompared to the extent of binding of hTg to said polyclonal humananti-hTg autoantibodies in said first reagent in the absence of saidtest sample, (B) determining the amount of said labeled reagentimmobilized on said solid phase by said immobilized reagent, and (C)correlating the amount of said labeled reagent immobilized by saidimmobilized reagent, if any, in the presence of test said sample withthe amount of said labeled reagent immobilized by said immobilizedreagent under the same conditions in the presence of a fluid referencesample containing a known amount of said hTg, whereby the amount of hTgautoantibody, if any, in said sample is determined.
 3. A methodaccording to claim 1 or claim 2, wherein said immobilized reagentcomprises said polyclonal human anti-hTg autoantibodies and said labeledreagent comprises human thyroglobulin.
 4. A method according to claim 1or claim 2, wherein said immobilized reagent comprises humanthyroglobulin and said labeled reagent comprises polyclonal humananti-hTg autoantibodies.
 5. A method according to claim 1 or claim 2,wherein said polyclonal human anti-hTg autoantibodies are provided inthe form of a serum obtained from one or more human subjects having athyroid autoimmune disease wherein serum anti-hTg autoantibodies areproduced.
 6. A method according to claim 5, wherein said polyclonalhuman anti-hTg autoantibodies are provided in the form of ananti-hTg-IgG fraction obtained by affinity purification from said serum.7. A method according to claim 6, wherein said affinity-purifiedanti-hTg-IgG preparation is obtained from said serum by specific bindingto and elution from hTg immobilized on a matrix.
 8. A method accordingto claim 1 or claim 2, wherein said solid phase is the inside wall of atest tube and said human anti-hTg autoantibodies are bound to saidinside wall of said test tube by a binder specific for human IgG.
 9. Akit for detection of anti-hTg autoantibodies in a fluid sample, whichkit comprises a first reagent comprising polyclonal human anti-hTgautoantibodies and a second reagent comprising humanthyroglobulin,wherein one of said first reagent and said second reagentis a labeled reagent and the other of said first reagent and said secondreagent is an immobilized reagent bound to a solid phase.
 10. A kitaccording to claim 9, wherein said first reagent comprises saidpolyclonal human anti-hTg autoantibodies comprise an anti-hTg-IgGfraction obtained by affinity purification obtained from serum of one ormore human subjects having a thyroid autoimmune disease wherein serumanti-hTg autoantibodies are produced, and said solid phase is the insidewall of a test tube.